Rapid clearance from the circulation, inactivation by proteases and inhibitors, and a lack of affinity for the desired target sites of action limit the utility of potent but labile therapeutic proteins (Muzykantov, V. R., J Control Release 2001, 71, (1), 1-21). Diverse drug delivery systems (e.g., natural lipoproteins, liposomes and polymer nanocarriers) are being widely designed in order to maximize drug efficacy and minimize side effects (Langer, R., Nature 1998, 392, (6679 Suppl), 5-10). For example, polyethylene glycol (PEG), a hydrophilic polymer that enhances aqueous solubility, masks drugs and carriers from host defense systems and prolongs circulation in the bloodstream (“stealth” technology) (Moghimi, S. M. et al, Prog Lipid Res 2003, 42, (6), 463-78; Roux, E. et al, Biomacromolecules 2003, 4, (2), 240-248). Nanocarriers coated with PEG are already in clinical use for the intravascular delivery of anti-tumor agents, in the form of stealth liposomes (e.g., Doxil®) (Lasic, D. D., Nature 1996, 380, (6574), 561).
Comparatively little success has been achieved, however, in nanocarrier mediated delivery of therapeutic proteins, which is especially challenging because protein's biological activity requires maintaining it's native folded state. Loading therapeutic proteins into biodegradable polymer nanocarriers (PNC) can be complicated by protein unfolding and inactivation. Loss of enzymatic activity due to protein unfolding in harsh conditions of PNC formulation has represented a major barrier to the use of biodegradable co-polymers for delivery of therapeutic enzymes.
Formulations based on synthetic amphiphilic copolymers that consist of hydrophobic blocks conjugated with hydrophilic PEG blocks yield a variety of aggregate shapes, namely micelles, vesicles and frozen particles—a versatile palette of polymer nanocarriers with further diversity in size and degradation patterns (Discher, D. E. et al, Science 2002, 297, (5583), 967-73; Zhang, J. et al., Biomacromolecules 2006, 7, (9), 2492-2500; Vinogradov, S. V., et al, Bioconjug Chem 1998, 9, (6), 805-12; Discher, D. E. et al, 2002, cited above; Ravenelle, F. et al, Biomacromolecules 2003, 4, (3), 856-858; Zhang, L.; Eisenberg, A., Science 1995, 268, (5218), 1728-1731; and Alakhov, V. Y. et al, Expert Opin Investig Drugs 1998, 7, (9), 1453-73). However, encapsulation of large therapeutic proteins, especially enzymes, without loss of their biological activity into these polymer nanoparticles formed by self-assembly mechanism has not been reported, because conditions providing their formulation via this mechanism are not compatible with retaining enzymatic activity.
A relatively mild freeze-thaw double emulsion method for the encapsulation of active catalase, a large 249 kDa tetrameric enzyme, into PEG-PL(G)A (poly lactic-co-glycolic acid) PNC is discussed in US Patent Application Publication No. 2006/0127386. PLGA is a biodegradable FDA-approved co-polymer used for the production of drug delivery systems and sutures. Furthermore, H2O2, a reactive oxygen species widely implicated in the pathogenesis of many disease conditions (Muzykantov 2001, cited above) is freely diffusible through PL(G)A (Dziubla, T. D.; et al, J Control Release 2005, 102, (2), 427-39). Catalase encapsulated within PEG-PL(G)A PNC as discussed in the preceding three publications was protected from proteolysis and decomposed H2O2 diffusing through the PNC shell. The freeze thaw cycle added during the primary emulsion enhanced catalase loading into PNC and reduced its formulation-induced inactivation.
Despite the specificity of therapeutic enzymes, medical utility is often limited by inadequate delivery and insufficient stability in the body. For example, catalase is a naturally occurring antioxidant enzyme that can be used for the treatment of vascular oxidative stress involved in the pathogenesis of many disease conditions (Muzykantov 2001, cited above). However, catalase and other antioxidant enzymes (e.g., superoxide dismutase) have no practical medical utility due to inadequate delivery to therapeutic sites, especially the endothelial cells lining the luminal surface of blood vessels. Conjugation of enzymes to targeting antibodies improves delivery and effects of antioxidant enzymes in diverse animal models (Christofidou-Solomidou, M. et al, Am J Physiol Lung Cell Mol Physiol 2003, 285, (2), L283-92; Kozower, B. D. et al, Nat Biotechnol 2003, 21, (4), 392-8), and yet therapeutic duration is limited to a few hours by catalase proteolysis at the target site (Muro, S. et al, Am J Physiol Cell Physiol 2003, 285, (5), C1339-47).
There remains a need in the art for improved compositions and methods for targeting active therapeutic proteins to cells which maintains folded and active protein, provides protection of the encapsulated proteins from subsequent proteolysis degradation, and prolongs their biological activity in vivo.